Bordetella pertussis is a Gram-negative coccobacillus that causes whooping cough or pertussis, a respiratory disease that has recently experienced a resurgence. Upon entering the respiratory tract, B. pertussis colonizes the airway epithelium and attaches to ciliated cells. Here, we used primary human nasal epithelial cells (hNECs) cultured at the air-liquid interface and investigated their interaction with B. pertussis B1917, focusing on the role of the type III secretion system effector protein BteA. In this model, which resembles the epithelial cells of nasal epithelium in vivo, B. pertussis B1917 localized predominantly in the overlying mucus and scarcely colonized the cell cilia. The colonization led to a gradual decline in epithelial barrier function, as shown by measurements of transepithelial electrical resistance (TEER) and staining of the tight junction protein zonula occludens 1. The decrease in TEER occurred independently of the cytotoxic effector protein BteA. Transcriptomic and proteomic analyses of hNECs showed only moderate changes following infection, primarily characterized by increased mucus production, including upregulation of mucin MUC5AC. No profound response to BteA was detected. Furthermore, the infection did not induce production of inflammatory cytokines, suggesting that B. pertussis B1917 evades recognition by hNECs in this model system. These results suggest that the mucus may serve as a niche that allows B. pertussis B1917 to minimize epithelial recognition and damage. The lack of a robust immune response further indicates that additional components of the nasal mucosa, such as innate immune cells, are likely required to initiate an effective host defense.IMPORTANCEThe nasal epithelium is the initial site where Bordetella pertussis comes into contact with the host during respiratory tract infection. In this study, human nasal epithelial cells cultured at the air-liquid interface were established as an in vitro model to investigate the early stages of B. pertussis infection. We showed that the clinical isolate B. pertussis B1917 resides in the mucus during the early stages of colonization without disrupting the epithelial barrier function. Infection results in moderate transcriptomic and proteomic changes, characterized by increased mucus production and minimal inflammatory signaling. These results suggest that B. pertussis B1917 may evade early host recognition by residing in mucus and avoiding direct interaction with epithelial cells. They also highlight the importance of other components of the mucosal immune system, such as resident immune cells, for the initiation of an effective defense.

• Keywords
• Bordetella pertussis, BteA effector, air-liquid interface culture, airway epithelium, human nasal epithelial cell, type III secretion system,
• MeSH
• Bacterial Proteins metabolism   genetics   MeSH
• Bordetella pertussis * pathogenicity   genetics   physiology   MeSH
• Epithelial Cells * microbiology   immunology   metabolism   MeSH
• Virulence Factors, Bordetella metabolism   MeSH
• Mucus microbiology   metabolism   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Mucin 5AC metabolism   genetics   MeSH
• Nasal Mucosa * microbiology   cytology   immunology   MeSH
• Whooping Cough * microbiology   immunology   MeSH
• Proteomics MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Virulence Factors, Bordetella MeSH
• Mucin 5AC MeSH

The type III secretion system (T3SS) is an important virulence factor of Gram-negative bacteria, including the genus Aeromonas, which represents a diverse group of aquatic bacteria. One member of the genus, Aeromonas schubertii, is an emerging pathogen in aquaculture, causing high mortality in snakehead fish. Infections are associated with the formation of white nodules in the internal organs, likely resulting from A. schubertii-induced apoptosis and/or necrosis. The present study investigates the type strain A. schubertii ATCC 43700, which encodes two distinct T3SSs located within Aeromonas pathogenicity islands 1 and 2, referred here to as API1 and API2. We analyzed their role in A. schubertii-induced cytotoxicity and identified novel T3SS effector proteins. Infections of HeLa cells revealed that API1, but not API2, mediates cytotoxicity and induces both apoptotic and necrotic cell death. Moreover, proteomic analysis identified seven candidate effectors secreted by the API1 injectisome. These included two previously described effectors, AopH and AopO from A. salmonicida, as well as five novel effectors named AopI, AopJ, AopL, AopT, and AopU, whose injection into host cells was validated using a split luciferase reporter system. Functional characterization showed that AopL, a homolog of Vibrio parahaemolyticus VopQ, induces caspase-3/-7-independent necrosis, while AopI, a homolog of ExoY from Pseudomonas aeruginosa, suppresses caspase-3/-7 activation and necrosis, revealing a pro-survival function. These results demonstrate the critical role of the API1 injectisome in A. schubertii-induced cytotoxicity and provide experimental identification of novel Aeromonas effectors that cooperate to fine-tune host cell cytotoxicity.

• Keywords
• Aeromonas, Aeromonas schubertii, ExoY, VopQ, cytotoxicity, type III secretion system effectors,
• MeSH
• Aeromonas * genetics   pathogenicity   physiology   MeSH
• Apoptosis MeSH
• Bacterial Proteins * metabolism   genetics   MeSH
• Virulence Factors * metabolism   genetics   MeSH
• Gram-Negative Bacterial Infections * microbiology   veterinary   MeSH
• HeLa Cells MeSH
• Humans MeSH
• Fish Diseases * microbiology   MeSH
• Type III Secretion Systems * metabolism   genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins * MeSH
• Virulence Factors * MeSH
• Type III Secretion Systems * MeSH

Bordetella pertussis is the causative agent of whooping cough in humans, a disease that has recently experienced a resurgence. In contrast, Bordetella bronchiseptica infects the respiratory tract of various mammalian species, causing a range of symptoms from asymptomatic chronic carriage to acute illness. Both pathogens utilize type III secretion system (T3SS) to deliver the effector protein BteA into host cells. Once injected, BteA triggers a cascade of events leading to caspase 1-independent necrosis through a mechanism that remains incompletely understood. We demonstrate that BteA-induced cell death is characterized by the fragmentation of the cellular endoplasmic reticulum and mitochondria, the formation of necrotic balloon-like protrusions, and plasma membrane permeabilization. Importantly, genome-wide CRISPR-Cas9 screen targeting 19,050 genes failed to identify any host factors required for BteA cytotoxicity, suggesting that BteA does not require a single nonessential host factor for its cytotoxicity. We further reveal that BteA triggers a rapid and sustained influx of calcium ions, which is associated with organelle fragmentation and plasma membrane permeabilization. The sustained elevation of cytosolic Ca2+ levels results in mitochondrial calcium overload, mitochondrial swelling, cristolysis, and loss of mitochondrial membrane potential. Inhibition of calcium channels with 2-APB delays both the Ca2+ influx and BteA-induced cell death. Our findings indicate that BteA exploits essential host processes and/or redundant pathways to disrupt calcium homeostasis and mitochondrial function, ultimately leading to host cell death.IMPORTANCEThe respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica exhibit cytotoxicity toward a variety of mammalian cells, which depends on the type III secretion effector BteA. Moreover, the increased virulence of B. bronchiseptica is associated with enhanced expression of T3SS and BteA. However, the molecular mechanism underlying BteA cytotoxicity is elusive. In this study, we performed a CRISPR-Cas9 screen, revealing that BteA-induced cell death depends on essential or redundant host processes. Additionally, we demonstrate that BteA disrupts calcium homeostasis, which leads to mitochondrial dysfunction and cell death. These findings contribute to closing the gap in our understanding of the signaling cascades targeted by BteA.

• Keywords
• Bordetella, calcium homeostasis, effector protein BteA, host cell death mechanism, type III secretion system (T3SS),
• MeSH
• Bacterial Proteins * metabolism   genetics   MeSH
• Bordetella bronchiseptica genetics   metabolism   drug effects   MeSH
• Bordetella pertussis genetics   pathogenicity   metabolism   drug effects   MeSH
• Cell Death * drug effects   MeSH
• Endoplasmic Reticulum metabolism   drug effects   MeSH
• Homeostasis * MeSH
• Host-Pathogen Interactions MeSH
• Humans MeSH
• Mitochondria metabolism   drug effects   MeSH
• Type III Secretion Systems metabolism   genetics   MeSH
• Calcium * metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins * MeSH
• Type III Secretion Systems MeSH
• Calcium * MeSH

Bordetella pertussis is a Gram-negative, strictly human re-emerging respiratory pathogen and the causative agent of whooping cough. Similar to other Gram-negative pathogens, B. pertussis produces the type III secretion system, but its role in the pathogenesis of B. pertussis is enigmatic and yet to be elucidated. Here, we combined RNA-seq, LC-MS/MS, and co-immunoprecipitation techniques to identify and characterize the novel CesT family T3SS chaperone BP2265. We show that this chaperone specifically interacts with the secreted T3SS regulator BtrA and represents the first non-flagellar chaperone required for the secretion of an anti-sigma factor. In its absence, secretion but not production of BtrA and most T3SS substrates is severely impaired. It appears that the role of BtrA in regulating T3SS extends beyond its activity as an antagonist of the sigma factor BtrS. Predictions made by artificial intelligence system AlphaFold support the chaperone function of BP2265 towards BtrA and outline the structural basis for the interaction of BtrA with its target BtrS. We propose to rename BP2265 to BtcB for the Bordetella type III chaperone of BtrA.In addition, the absence of the BtcB chaperone results in increased expression of numerous flagellar genes and several virulence genes. While increased production of flagellar proteins and intimin BipA translated into increased biofilm formation by the mutant, enhanced production of virulence factors resulted in increased cytotoxicity towards human macrophages. We hypothesize that these phenotypic traits result indirectly from impaired secretion of BtrA and altered activity of the BtrA/BtrS regulatory node.

• Keywords
• Bordetella pertussis, CesT chaperone, T3SS, anti-sigma factor, biofilm,
• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Bordetella pertussis * metabolism   MeSH
• Chromatography, Liquid MeSH
• Humans MeSH
• Whooping Cough * MeSH
• Gene Expression Regulation, Bacterial MeSH
• Sigma Factor genetics   MeSH
• Tandem Mass Spectrometry MeSH
• Artificial Intelligence MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Sigma Factor MeSH